1. Field of the Invention
The present invention relates to a method of spotting a probe on a solid support, a probe array and a method of manufacturing thereof, and a method of detecting a target single-stranded (ss) nucleic acid and a method of identifying a base sequence of a target ss nucleic acid using the probe array.
2. Related Background Art
As a method to determine a base sequence of a nucleic acid, detect a target nucleic acid in a sample, and identify various bacteria swiftly and accurately, proposed is the use of a probe array where one or more substances which can bind specifically to a target nucleic acid, so-called probes, are arranged on a solid support at a large number of sites. As a general method of manufacturing such probe arrays as described in EP No. 0373203B1, (1) the nucleic acid probe is synthesized on a solid support or (2) a previously synthesized probe is supplied onto a solid support. U.S. Pat. No. 5,405,783 discloses the method (1) in detail. Concerning the method (2), U.S. Pat. No. 5,601,980 and Science Vol. 270, p. 467 (1995) teach a method of arranging cDNA in an array by using a micropipet.
In the above method (1), it is not necessary to synthesize a nucleic acid probe in advance, since the nucleic acid probe is synthesized directly on a solid support. However, it is difficult to purify a probe nucleic acid synthesized on a solid support. The accuracy in determining the base sequence of a nucleic acid and in the detection of a target nucleic acid in a sample using a probe array largely depends on the correctness of the base sequence of the nucleic acid probe. For the method (1), therefore, further improvement in accuracy of a nucleic acid probe is required in order to manufacture a probe array of higher quality. In the method (2), a step of synthesizing a nucleic acid probe is required prior to the fixation of the nucleic acid probe on a solid support, but the nucleic acid probe can be purified before binding the probe to a solid support. For this reason, presently, the method (2) is considered to be more preferable than the method (1) as a method of manufacturing a probe array of high quality. However, the method (2) has a problem in the method of spotting a nucleic acid probe densely on a solid support. For example, when a probe array is used to determine the base sequence of a nucleic acid, it is preferable to arrange as many kinds of nucleic acid probes as possible on a solid support. When mutations in a gene are to be detected efficiently, it is preferable to arrange nucleic acid probes of sequences corresponding to the respective mutations on a solid support. In addition, when a target nucleic acid in a sample or to gene mutations and deletions are detected, it is desirable that the amount of the sample taken from a subject, specifically a blood sample, is as small as possible. Thus, it is preferable that as much information as possible on the base sequence is obtained using a small sample amount. Considering these points, it is preferable that, for example, 10,000 or more nucleic acid probe spots per square inch are arranged in a probe array.